Startseite Medizin Post-transcriptional regulation of miRNA-15a and miRNA-15b on VEGFR gene and deer antler cell proliferation
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Post-transcriptional regulation of miRNA-15a and miRNA-15b on VEGFR gene and deer antler cell proliferation

  • Mingxiao Liu ORCID logo , Xiangyu Han , Dongming Cui , Yuduo Yan , Lu Li und Wei Hu EMAIL logo
Veröffentlicht/Copyright: 16. November 2018
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Turkish Journal of Biochemistry
Aus der Zeitschrift Turkish Journal of Biochemistry Band 44 Heft 3

Abstract

Background

Deer antler is the only regenerative organ in mammals, the regeneration of antler is not only the regeneration of bone tissue, but also accompanied by the regeneration of nerves, blood vessels and so on. The purpose of the current study was to explore the effect of miRNA-15a and miRNA-15b on the regulation of sika deer vascular endothelial growth factor receptor (VEGFR) during rapid antler growth.

Materials and methods

The VEGFR 3′-UTR was analyzed by bioinformatics software to identify the highly matched miRNAs. After transfected with miRNA mimics, the expression of selected miRNAs were measured by RT-qPCR and the relative expression level of VEGFR protein was detected by Western Blot. Dual-luciferase activity assay was used to determine the target relationship between VEGFR and miRNAs. The cartilage cell proliferation and telomerase activity were measured by MTT kit and TRAP assay, respectively.

Results

The VEGFR 3′-UTR contains a binding site for miRNA-15a and miRNA-15b. Over-expression of miRNA-15a and miRNA-15b, which significantly reduced the expression level of VEGFR protein, inhibited the proliferation of cartilage cells, and decreased the telomerase activity of cartilage cells in vitro.

Conclusion

miRNA-15a and miRNA-15b represent novel regulatory factors of VEGFR expression in deer antler.

Öz

Amaç

Geyik boynuzu memelilerdeki tek rejeneratif organdır, boynuzun rejenerasyonu sadece kemik dokusunun rejenerasyonu değildir, aynı zamanda sinirlerin, kan damarlarının vb. Bu çalışmanın amacı, miRNA-15a ve miRNA-15b′nin hızlı boynuz büyümesi sırasında, sika geyiği vasküler endotelyal büyüme faktörü reseptörünün (VEGFR) düzenlenmesi üzerindeki etkisini araştırmaktır.

Gereç ve yöntem

VEGFR 3′-UTR, yüksek uyumlu miRNA’ları tanımlamak için biyoinformatik yazılımı ile analiz edildi. miRNA taklitleri ile transfeksiyondan sonra, seçilmiş miRNA’ların ifadesi RT-qPCR ile ölçüldü ve VEGFR proteinlerinin nispi ekspresyon seviyeleri Western Blot tarafından tespit edildi. VEGFR ve miRNA’lar arasındaki hedef ilişkiyi belirlemek için çift-lusiferaz aktivitesi analizi kullanıldı. Kıkırdak hücre proliferasyonu ve telomeraz aktivitesi, sırasıyla MTT kiti ve TRAP testi ile ölçüldü.

Bulgular

VEGFR 3′-UTR, miRNA-15a ve miRNA-15b için bir bağlanma bölgesi içerir. VEGFR proteininin ekspresyon seviyesini önemli ölçüde azaltan miRNA-15a ve miRNA-15b′nin aşırı ekspresyonu, in vitro ortamda kıkırdak hücrelerinin proliferasyonunu inhibe etti ve kıkırdak hücrelerinin telomeraz aktivitesini düşürdü.

Sonuç

miRNA-15a ve miRNA-15b, geyik boynuzunda VEGFR ifadesinin yeni düzenleyici faktörlerini temsil etmektedir.

Keywords: Sika deer; Vascular endothelial growth factor receptor; miRNA-15a; miRNA-15b; Telomerase activity
Anahtar Kelimeler: Sika geyiği; Vasküler endotelyal büyüme faktörü reseptörü; miRNA-15a; miRNA-15b; Telomeraz aktivitesi

Introduction

Antlers are a secondary sexual characteristic of male sika deer and are currently known to be the only mammalian appendant organ that can undergo complete regeneration upon loss [1]. The regeneration of deer antler is based on the process of stem cell aggregation in the pedicle periosteum [2]. As a special bone tissue, the deer antler growth center is located in the tip region, and it has been speculated that this region contains active substances which differ from those found in general tissue and stimulate deer antler growth [3]. Therefore, deer antler is an ideal model for investigating wound repair and organ regeneration in mammals. Sika deer antler contains an extreme abundance of blood vessels that distribute the large amounts of nutrients needed for rapid antler growth. There are many cellular growth factors involved in angiogenesis, of which vascular endothelial growth factor (VEGF) is a functional glycoprotein with high biological activity that acts on vascular endothelial cells to specifically promote endothelial cell growth, hematopoietic stem cell division, and venule proliferation [4], [5]. Additionally, VEGF induces angiogenesis and increases capillary permeability [6]. VEGF induces angiogenesis and increases vascular permeability through binding to VEGFR on vascular endothelial cells [7], which stimulates the accumulation of inositol triphosphate and simultaneously causes calcium influx and increases intracellular calcium concentration by more than three-fold within a short time [8]. Increased vascular permeability favors macromolecular protein extravasation and the formation of fibrinogen to supply vascular endothelial cell growth. At present, VEGFR are divided into two classes: tyrosinase and non-tyrosinase receptors, of which tyrosinase receptors, such as fms related tyrosine kinase-1 (Flt-1), kinase insert domain receptor (KDR), fms related tyrosine kinase-3 (Flt-3), have high affinity for VEGF [9]. Previous studies have found that VEGF binds to KDR, induces receptor dimerization, stimulates receptor tyrosine phosphorylation, and activates downstream signaling [10]. In relation to VEGF signaling in vascular endothelial cells, the interaction between VEGF and KDR is crucial for proliferation and differentiation. We therefore prioritized our research on KDR signaling.

Gene regulation is a dynamic multi-channel, multi-level, and multi-mode process, the transcriptional and post-transcriptional regulation is of great biological importance. In such regulation processes, RNAs (microRNAs or miRNAs) play important roles in molecular regulation [11]. MiRNAs can bind to the 3′-UTR of target mRNAs and negatively regulate post-transcriptional levels through degradation, via the miRNA-induced silencing complex, or direct inhibition of protein translation [12]. Many studies have demonstrated that miRNAs play vital roles in the pathogenesis of vascular diseases, however the mechanisms through which specific miRNAs regulate angiogenesis are poorly understood.

The miRNA-15 family includes miRNA-15a, miRNA-15b, miRNA-16, miRNA-195, miRNA-424, miRNA-497, and others, of which miRNA-15a and miRNA-16 are expressed in all vertebrates [13], [14]. In the miRNA-15 family, miRNA-15a/16-1 and miRNA-15b/16-2 are two gene clusters, of which the sequence of miRNA-16-1 is identical to the sequence of miRNA-16-2, the miRNA-15a and miRNA-15b differ by four nucleotides and have identical seed region sequences [15]. The sequences of miRNA-15a/16-1 and miRNA-15b/16-2 are highly conserved in rats, mice, rhesus monkeys, humans, and other mammals [16]. Studies have shown that miRNA-15b targets the VEGFR gene in human vascular endothelial cells, and over-expression of miRNA-15b inhibits VEGFR protein expression and affects human vascular endothelial cell migration [17], furthermore, injections of the pre-miRNA-15b precursor into zebrafish significantly inhibit small intestine vascularisation [18]. Changes in miRNA-15a and miRNA-15b expression have been implicated in the occurrence and development of many diseases, including cancer, cardiovascular disease, and neurodegenerative disease. Therefore, the effects of miRNA-15a and miRNA-15b on signal transduction pathways are regarded as immensely important by many scholars. Based on the above, our research aims to investigate the effect of miRNA-15a and miRNA-15b on the regulation of sika deer VEGFR during rapid antler growth, and the effect on proliferation of antler cartilage cells via overexpressing miRNA-15a and miRNA-15b.

Materials and methods

Selection of potential VEGFR-targeting miRNAs and analysis of miRNA binding sites in deer antler cells

The VEGFR 3′-UTR was analyzed using bioinformatics software TargetScan 6.2 and miRanda to identify matched and highly conserved miRNAs [19]. Combined with data from the antler apical tissue miRNA genechip previously prepared in our lab, we selected miRNAs capable of regulating VEGFR from those differentially expressed in deer antler cartilage and mesenchymal tissues. Meanwhile, we analyzed the VEGFR 3′-UTR to identify the potential region that interacts with miRNA-15a and miRNA-15b.

Cell culture

Deer antler was obtained from a 3-year-old male sika deer (Cervus nippon) provided by Jilin Agricultural University Deer Farm (Changchun, China). At first, the cartilage tissue was isolated under a dissecting microscope and cut it into small pieces of about 1 mm3, then the culture of cartilage cells were conducted following the protocol as described [20]. The tissues were digested by collagenase-I and hyaluronidase for 1.5 h at 37°C, after which they were digested by collagenase-II for 3 h under the same conditions. After centrifugation, the cartilage cells were cultured in dulbecco’s modified eagle medium (DMEM) supplemented with 20% (v/v) fetal bovine serum, 200 U/mL penicillin and 100 U/mL streptomycin at 37°C with 5% (v/v) CO2. The study was approved by the ethics committee of the Institute of Life Sciences of Jilin Agriculture University in Changchun, China.

Cell transfection

Cartilage cells were divided into three groups: untreated group, negative control group (cells transfected with a miRNA negative control mimics), and experimental group (cells transfected with miRNA-15a or miRNA-15b mimics). When the cells reached a growth density of more than 80%, the cells were transfected using Roche HP transfection reagent according to the manufactures’ protocols. The procedure is as follows: add 200 μL DMEM medium to two RNase-free EP tubes, add 5 μL miRNA mimics and transfection reagent, respectively. After 5 min at room temperature, the two were mixed and incubated for 20 min at room temperature. Subsequently, the transfection complex was added dropwise to the cells and cultured overnight under 5% (v/v) CO2 at 37°C.

Detection of miRNA-15a and miRNA-15b expression level

Cartilage cells were trypsinized and transferred into six-well plates at a density of 106 cells/well by cell counting. At 24 h, 48 h, and 72 h after transfection with miRNA mimics, cells were collected, and the expression of selected miRNAs was measured by RT-qPCR (SYBR Green I dye method) with U6 as an internal control. Each experiment was repeated in triplicate, and the relative expression levels of miRNAs were calculated using the 2−ΔΔCt method.

Dual-luciferase activity assay

The 3′-UTR of VEGFR and its mutant (mutation the seed region recognized by miRNA-15a/15b on the VEGFR 3′-UTR) were cloned by Sunbiotech Co. Ltd. (Beijing), after which the VEGFR 3′-UTR and its mutant were inserted into the dual-luciferase reporter vector (pmiR-Report™) to construct wild-type/mutant-type VEGFR 3′-UTR dual-luciferase reporter vector, respectively. Then, cartilage cells at a density of 106 cells/well were seeded into six-well plates and incubated overnight. When the growth density reached 70%, cells were co-transfected with pmiR-Report-VEGFR-3′-UTR (wild-type/mutant-type) and miRNA-15a or miRNA-15b mimics. The luciferase activity in each group was measured at 24 h, 48 h, and 72 h after transfection, using the Dual-Luciferase Reporter Assay Kit (Promega, USA).

Western Blot analysis

Cartilage cells were harvested at 24 h, 48 h, and 72 h after transfection, and total protein was extracted using RIPA lysis buffer with 1 mM PMSF at 0°C. Then, the Western Blot analysis were performed as described [21]. Protein samples were separated by 12% SDS-PAGE and electrophoretically transferred to PVDF membranes. The membranes were blocked with 5% evaporated milk, and then the membranes were incubated with primary antibodies of VEGFR and GAPDH (Bioss, Beijing, China) overnight at 4°C. After that, the membranes were incubated with the secondary antibodies at room temperature for 2 h. After washing with TBST three times, the signals were detected using Enhanced Chemi-luminescence reagents and the protein band intensities were analyzed using Image-Pro plus software.

Cell proliferation assay

Cartilage cells at a density of 4000 cells/well were seeded into 96-well plates and incubated overnight. After transfect for 24 h, 48 h, and 72 h, cell growth was measured using MTT method as described in our previous work [20]. In brief, cell cultural medium was replaced with 0.1 mL fresh medium containing 0.5 mg/mL MTT. Cells were incubated at 37°C for 4 h, after which the bluish-violet crystalline precipitate was dissolved in 0.1 mL DMSO. The absorbance was measured at 490 nm.

Determination of telomerase activity by the TRAP assay

Telomeric repeat amplification protocol (TRAP) was carried out as previously described [22]. First, the cartilage cells were collected at 24 h, 48 h and 72 h after transfection, and then we added 200 μL of CHAPS lysate (pre-added with PMSF and mercaptoethanol) to the cell sediment. After lysis on ice for 30 min, they were centrifuged at 4°C, 12,000 r/min for 30 min, and the supernatant was taken as a template. The TRAP primers designed by C language and validated by the DNAStar software were as follows: P1: 5′-TCACCTTGTAATCCGTCGAGCAGAGTT-3′, P2: 5′-CAACATCTCCACTACCTTCTAACCGTAACC-3′, and the primers were synthesized by Takara Biotechnology Co. Ltd. (Dalian, China). The P1 primer (27 nt) was consisted of the telomerase strand at the 3′ terminal (18 nt) and random sequence at the 5′ terminal (9 nt). The P2 primer (30 nt) had the telomere binding region (12 nt) at the 3′ terminal and the anchoring sequence (18 nt) at the 5′ terminal.

PCR reaction system contained 5 μL of 10×TRAP buffer (200 mM Tris-HCl pH 8.3, 15 mM MgCl2, 1 mL/L Tween-20, 630 mM KCl, 10 mM EDTA), 50 μM of dATP, dTTP, dGTP and dCTP (add 2 μL 1.25 mM dCTP to the PCR tube, and then quickly add 10 μL thawed paraffin of dCTP), 15 pmol/μL of P1 and P2 primers, 2 μL of Taq polymerase and 2 μL of template, the reaction volume was made up to 50 μL by adding sterile DEPC. For telomerase reaction, the EP tube was incubated at 25°C for 20 min to synthesize the telomere template and then heated at 90°C for 3 min to inactive the telomerase. Then, the PCR reaction condition was 90°C for 30 s, 64°C for 45 s, 72°C for 1 min and 30 cycles. The PCR products were analyzed by 12% non-denaturing SDS-PAGE with silver staining. Then, the gel was scanned into Tiff-format image, and the bands were analyzed by Bandscan software for assessment of the band density and the relative telomerase activity.

Statistical analysis

All data was presented as means±standard deviation (S.D.). SPSS Statistics 12.0 software was used for all the statistical analysis and p-values<0.05 was considered to be statistically significant.

Results

Primary selection of miRNAs that regulate VEGFR in deer antler cells

TargetScan and miRanda were used to analyze the VEGFR 3′-UTR in deer antler cells. As a result, 31 miRNAs were selected that were highly conserved among species and contained the incomplete binding sequence of the VEGFR 3′-UTR. These miRNAs may decrease VEGFR expression. We combined this data with results from our antler cartilage and mesenchymal tissue miRNA genechips, and subsequently selected two differentially expressed miRNAs, miRNA-15a and miRNA-15b (Figure 1), for further analysis.

Figure 1: Conservative analysis of the miRNA binding sites in the 3′-UTR of VEGFR.In Chinese sika deer antler, VEGFR may be a molecular target of miRNA-15a and miRNA-15b. This figure shows the seed region sequence of miRNA-15a and miRNA-15b and its conserved target site in the 3′-UTR of VEGFR, which was downloaded from TargetScan. As predicted, two sections of miRNA-15a and miRNA-15b bind to the 3′-UTR of VEGFR, respectively.
Figure 1:

Conservative analysis of the miRNA binding sites in the 3′-UTR of VEGFR.

In Chinese sika deer antler, VEGFR may be a molecular target of miRNA-15a and miRNA-15b. This figure shows the seed region sequence of miRNA-15a and miRNA-15b and its conserved target site in the 3′-UTR of VEGFR, which was downloaded from TargetScan. As predicted, two sections of miRNA-15a and miRNA-15b bind to the 3′-UTR of VEGFR, respectively.

Detection of miRNA-15a and miRNA-15b expression levels by RT-qPCR

The expression levels of miRNA-15a and miRNA-15b in cartilage cells were determined by RT-qPCR analysis. The expression levels of miRNA-15a and miRNA-15b in transfected cells significantly increased in comparison with untreated cells. As shown, miRNA-15a (Figure 2A) and miRNA-15b (Figure 2B) expression levels were higher at 48 h after transient transfection, compared to 24 h and 72 h. This indicated that we successfully transfected miRNA-15a and miRNA-15b into cartilage cells.

Figure 2: The relative quantitative results for the content of miRNA-15a and miRNA-15b.The relative quantitative results of (A) miRNA-15a and (B) miRNA-15b in sika deer antler cartilage cells after transfection for 24 h, 48 h and 72 h were shown. The U6 gene was used as a loading control. The content of miRNA-15a and miRNA-15b in the cartilage cells increased markedly compared with the untreated group and negative control group. Data are presented as the mean±S.D., n=3. *p<0.05, **p<0.01 vs. untreated group.
Figure 2:

The relative quantitative results for the content of miRNA-15a and miRNA-15b.

The relative quantitative results of (A) miRNA-15a and (B) miRNA-15b in sika deer antler cartilage cells after transfection for 24 h, 48 h and 72 h were shown. The U6 gene was used as a loading control. The content of miRNA-15a and miRNA-15b in the cartilage cells increased markedly compared with the untreated group and negative control group. Data are presented as the mean±S.D., n=3. *p<0.05, **p<0.01 vs. untreated group.

Dual-luciferase activity assay

The wild-type or mutant-type pmiR-Report-VEGFR-3′-UTR plasmids were co-transfected with miRNA-15a or miRNA-15b mimics into cartilage cells, respectively. Then, the cells were harvested at 24 h, 48 h and 72 h. Luciferase activity was measured to investigate whether miRNA-15a and miRNA-15b regulate VEGFR protein expression through targeting the VEGFR 3′-UTR. Our results showed (Figure 3), compared to the negative control group, that the relative luciferase activity significantly decreased, especially at 48 h, in cartilage cells transfected with pmiR-Report-VEGFR-3′UTR (wild-type) and miRNA mimics. However, after transfection with pmiR-Report-VEGFR-3′UTR (mutant-type) and miRNA mimics, the relative luciferase activity did not significantly changed in comparison to the negative control group. These data suggest that the VEGFR 3′-UTR contains a binding site for miRNA-15a and miRNA-15b, our transfection system was stable and effective, and the miRNAs, to a certain extent, inhibit the expression of proteins that contain this binding site.

Figure 3: VEGFR is a direct target of miRNA-15a and miRNA-15b.Sika deer antler cartilage cells were co-transfected with the Wild-type/mutant-type VEGFR luciferase reporter plasmids and miRNA-15a/miRNA-15b mimics, as indicated. Following 24 h, 48 h and 72 h, the luciferase activity was measured. Luciferase activity was decreased compared with the negative control group when cells were transfected with wild-type plasmids and mimics of miRNA-15a and miRNA-15b. After transfection with mutant-type plasmids and miRNA mimics, the relative luciferase activity did not significantly changed in comparison to the negative control group. Data are presented as the mean±S.D., n=3. *p<0.05, **p<0.01 vs. negative group.
Figure 3:

VEGFR is a direct target of miRNA-15a and miRNA-15b.

Sika deer antler cartilage cells were co-transfected with the Wild-type/mutant-type VEGFR luciferase reporter plasmids and miRNA-15a/miRNA-15b mimics, as indicated. Following 24 h, 48 h and 72 h, the luciferase activity was measured. Luciferase activity was decreased compared with the negative control group when cells were transfected with wild-type plasmids and mimics of miRNA-15a and miRNA-15b. After transfection with mutant-type plasmids and miRNA mimics, the relative luciferase activity did not significantly changed in comparison to the negative control group. Data are presented as the mean±S.D., n=3. *p<0.05, **p<0.01 vs. negative group.

Detection of VEGFR expression by Western Blot

Western Blot showed that VEGFR expression was decreased, in comparison with untreated cells, following transfection of antler cartilage cells with miRNA-15a and miRNA-15b mimics. The effects increased in a time-dependent manner (Figure 4). The analysis of densitometric data was consistent with the results of electrophoresis (Table 1).

Figure 4: Detection of the effect of miRNA mimics on the expression of VEGFR protein.When cells were transfected with miRNA-15a and miRNA-15b mimics for 24 h, 48 h and 72 h, the expression of VEGFR protein and GAPDH in sika deer cartilage cells was detected using Western Blot analysis. The protein levels of VEGFR were decreased compared to the untreated group, which indicated that miRNA-15a and miRNA-15b inhibited the expression of the VEGFR protein.
Figure 4:

Detection of the effect of miRNA mimics on the expression of VEGFR protein.

When cells were transfected with miRNA-15a and miRNA-15b mimics for 24 h, 48 h and 72 h, the expression of VEGFR protein and GAPDH in sika deer cartilage cells was detected using Western Blot analysis. The protein levels of VEGFR were decreased compared to the untreated group, which indicated that miRNA-15a and miRNA-15b inhibited the expression of the VEGFR protein.

Table 1:

Analysis of the expression intensity of VEGFR protein.

Group24 h after transfection48 h after transfection72 h after transfection
Untreated568462457
Negative control318308305
miR-15a mimic381229116
miR-15b mimic41111652
GAPDH10271165946

The effect of miRNA-15a and miRNA-15b on cartilage cells proliferation

MTT assay was used to determine whether miRNA-15a and miRNA-15b affect the proliferation of cartilage cells. Compared with the untreated group, cartilage cells exhibited significantly decreased proliferation at 48 h after miRNA-15a and miRNA-15b mimic transfection. These observations indicate that the antler cartilage cells proliferation could be regulated by miRNA-15a and miRNA-15b (Figure 5). This result also demonstrates that miRNA-15a and miRNA-15b act to inhibit antler cartilage cells proliferation.

Figure 5: The proliferation of cartilage cells was inhibited by miRNA-15a and miRNA-15b.Cartilage cells were transfected with negative control mimic or miRNA-15a/miRNA-15b mimic. Compared with the untreated group and negative control group, cell proliferation was inhibited when cells were transfected with miRNA-15a and miRNA-15b mimics for 24 h, 48 h and 72 h as indicated. Data are presented as the mean±S.D., n=6. *p<0.05, **p<0.01 vs. untreated group.
Figure 5:

The proliferation of cartilage cells was inhibited by miRNA-15a and miRNA-15b.

Cartilage cells were transfected with negative control mimic or miRNA-15a/miRNA-15b mimic. Compared with the untreated group and negative control group, cell proliferation was inhibited when cells were transfected with miRNA-15a and miRNA-15b mimics for 24 h, 48 h and 72 h as indicated. Data are presented as the mean±S.D., n=6. *p<0.05, **p<0.01 vs. untreated group.

TRAP assay of telomerase activity

Antler cartilage cells were transfected with miRNA-15a and miRNA-15b mimics. Cells were collected at 24 h, 48 h, and 72 h after transfection and TRAP assay was subjected to determine changes in telomerase activity (Figure 6). Compared with the untreated group, we found that telomerase activity was reduced in antler cartilage cells at 24 h, 48 h, and 72 h after transfection (Table 2). These results suggest that the telomerase activity may be related to the expression level of VEGFR.

Figure 6: TRAP assay detects the changes of telomerase activity in cartilage cells.Compared with the untreated group and negative control group, telomerase activity was reduced in deer antler cartilage cells at 24 h, 48 h, and 72 h after transfection, which indicated that telomerase activity may be related to the expression level of VEGFR. PC, Positive control; NC, Negative control.
Figure 6:

TRAP assay detects the changes of telomerase activity in cartilage cells.

Compared with the untreated group and negative control group, telomerase activity was reduced in deer antler cartilage cells at 24 h, 48 h, and 72 h after transfection, which indicated that telomerase activity may be related to the expression level of VEGFR. PC, Positive control; NC, Negative control.

Table 2:

Band density analysis of telomerase activity.

GroupUntreatedmiRNA-15amiRNA-15b
NC24 h48 h72 hNC24 h48 h72 h
Sum479.19372.13370.23319.12302.54439.82428.68357.51296.45

Discussion

In recent years, the molecular mechanisms of sika deer antler periodic regeneration and development have been widely investigated by scholars at home and abroad, and the powerful proliferative ability of tissue cells in the top region of antler makes sika deer antler an ideal medical model for studying organ regeneration and cancer treatment [23]. The regeneration of deer antler is a process that involves stem cells. The first antler tissue is derived from the proliferation and differentiation of antlerogenic periosteum (AP) stem cells, a class of stem cells that retain embryonic stem cell activity, in antler generation region [24]. After the first antler undergoes exfoliation, the pedicle periosteum (PP) on frontal bone of sika deer permanently remains, AP disappears, and raw antler stem cells preserved in the PP differentiate and regenerate to form a complete antler the following year [25]. Activation of AP and PP cells is primarily regulated by androgens, which stimulate these cells to produce a large quantity of cell growth factors that stimulate rapid antler growth [26]. Antler bone tissue contains a remarkably rich vascular system, which continually supplies the large amount of nutrients required for the rapid antler growth process [27]. The VEGF pathway is now recognized as the most efficient and rapid angiogenesis promoting factor [28], and is crucial for the initiation of angiogenesis.

Previous studies have shown that miRNAs coordinate antler cell proliferation by regulating growth factor expression in antler cells [21], [29]. It has been discovered that miRNA expression can be detected in circulating blood and tissues, which suggests that miRNA may play a role in vessel occurrence and development. The miRNA-15 family is highly conserved in mammals [30], [31], and researchers initially discovered that miRNA-15a is an oncogenesis-related miRNA in chronic lymphocytic leukemia [32]. It has also been shown that miRNA-15b, which is highly homologous to miRNA-15a, plays a role in angiogenesis-related VEGFR synthesis [33], and therefore, we hypothesized that miRNA-15a and miRNA-15b may play a role in the regulation of tumor cell angiogenesis through interaction with VEGFR. However, no studies on miRNA-15a and miRNA-15b in sika deer have been reported to date.

Our research began with the results of a previously prepared antler apical tissue miRNA genechip analysis, which identified the differential miRNA-15a and miRNA-15b expression in different antler tissues and suggested that the two miRNAs may regulate the VEGFR gene in sika deer. We found that binding sites of miRNA-15a and miRNA-15b likely exist in the VEGFR 3′-UTR through bioinformatics predictions, and further analysis with TargetScan and miRanda indicated that the two miRNAs may regulate VEGFR protein expression. In order to experimentally investigate these predictions, we generated an artificial, synthetic VEGFR 3′-UTR sequence and verified that VEGFR is regulated by miRNA-15a and miRNA-15b using a luciferase activity assay. We subsequently transfected synthetic miRNA-15a and miRNA-15b mimics into antler cartilage cells and investigated miRNA over-expressing at different times after transfection through RT-qPCR. The results of this analysis revealed that exogenously transfected miRNA-15a and miRNA-15b can be stably and efficiently expressed in cartilage cells and indicated that these miRNAs truly mimic endogenous miRNAs. Western Blot and MTT showed, in comparison with the untreated group, that cells transfected with miRNA-15a and miRNA-15b could decrease the expression of VEGFR protein and inhibit the proliferation of cartilage cells, in a time-dependent manner. These results further demonstrate that miRNA-15a and miRNA-15b can regulate VEGFR protein expression. Since VEGFR is an important receptor of VEGF, when the expression of VEGFR is inhibited, this protein can not form a heterodimer and stimulate downstream signaling. In this way, the ability of miRNAs to impede the VEGF pathway inhibits the proliferation of antler cartilage cells.

Telomerase is a reverse transcriptase with a special function, which is mainly composed of telomere RNA (TR) and reverse transcriptase (TERT) [34]. In tumor cells, stem cells and other cells with high proliferation and division, telomerase activity is highly active [35], [36]. It is speculated that telomerase activity may be closely related to cell proliferation and differentiation. In our previous studies, the telomerase activity was detected on skin layer, mesenchymal layer and cartilage layer from the antler tip tissue, respectively. Among them, the telomerase activity of mesenchymal layer is the highest, the cartilage layer is the higher and the skin layer is the lowest. Li’s [37] research showed that when the antler cartilage cells were transfected with miRNA-93-5p and miRNA-20b-5p mimics, the proliferation of cells was inhibited and the relative expression of VEGF proteins was also decreased; Then, the content of telomerase after transfection was measured by ELISA, the content of telomerase gradually reduced with the extension of time, compared with the control group. Our results of TRAP assay also showed that the telomerase activity of cells in the transfected group gradually decreased, which indicates that telomerase activity may be related to the expression level of VEGFR. This result is consistent with our previous research. However, the specific mechanism still needs to be further studied.

In conclusion, our research demonstrated that miRNA-15a and miRNA-15b could reduce the expression of VEGFR by post-transcriptional regulation, which in turn inhibited the proliferation of cartilage cells in sika deer antler tissue, meanwhile, we speculated that the inhibition of cells proliferation may be associated with the decrease of telomerase activity. This is the first study to explore the role of miRNA-15a and miRNA-15b in the regulation of VEGFR in antler cells, however, the specific mechanism through which these miRNAs mediate antler regeneration remains uncertain. It is necessary for scholars at home and abroad to further study the molecular pathways that mediate deer antler regeneration.

Acknowledgements

This study was supported by a grant from the National Natural Science Foundation of China (no. 31572372 and 30972083).

  1. Conflict of interest: There are no conflicts of interest among the authors.

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Received: 2018-04-26
Accepted: 2018-09-04
Published Online: 2018-11-16
Published in Print: 2019-05-01

©2019 Walter de Gruyter GmbH, Berlin/Boston

Artikel in diesem Heft

  1. Frontmatter
  2. Short Communication
  3. Acetone-water mixture is a competent solvent to extract phenolics and antioxidants from four organs of Eucalyptus camaldulensis
  4. Research Articles
  5. Proteases from Calotropis gigantea stem, leaf and calli as milk coagulant source
  6. A new method to quantify atmospheric Poaceae pollen DNA based on the trnT-F cpDNA region
  7. Expression of a functional recombinant vascular endothelial growth factor 165 (VEGF165) in Arabidopsis thaliana
  8. Computational exploration of antiviral activity of phytochemicals against NS2B/NS3 proteases from dengue virus
  9. Investigation of antioxidant, cytotoxic, tyrosinase inhibitory activities, and phenolic profiles of green, white, and black teas
  10. DFR and PAL gene transcription and their correlation with anthocyanin accumulation in Rhodomyrtus tomentosa (Aiton.) Hassk.
  11. Comparison of phenolic profiles and antioxidant activity of three Ornithogalum L. species
  12. Increasing the fermentation efficiency of Lactobacillus paracasei ssp. paracasei MIUG BL6 in a rye flour sourdough
  13. Determination of chemical composition, antibacterial and antioxidant properties of products obtained from carob and honey locust
  14. Chitinolytic Bacillus subtilis Ege-B-1.19 as a biocontrol agent against mycotoxigenic and phytopathogenic fungi
  15. Recycling fish skin for utilization in food industry as an effective emulsifier and foam stabilizing agent
  16. A novel, thermotolerant, extracellular PHB depolymerase producer Paenibacillus alvei PHB28 for bioremediation of biodegradable plastics
  17. Post-transcriptional regulation of miRNA-15a and miRNA-15b on VEGFR gene and deer antler cell proliferation
  18. Comparison of pendimethalin binding properties of serum albumins from various mammalian species
  19. Crocin (active constituent of saffron) improves CCl4-induced liver damage by modulating oxidative stress in rats
  20. Time dependent change of ethanol consumption biomarkers, ethyl glucuronide and ethyl sulphate, after single dose ethanol intake
  21. GC-MS analysis and biological activities of Thymus vulgaris and Mentha arvensis essential oil
  22. Immobilization and some application of α-amylase purified from Rhizoctonia solani AG-4 strain ZB-34
  23. Letter to the Editor
  24. Molecular crosstalk between Hog1 and calcium/CaM signaling
https://doi.org/10.1515/tjb-2018-0160
Schlagwörter für diesen Artikel
Sika deer; Vascular endothelial growth factor receptor; miRNA-15a; miRNA-15b; Telomerase activity

Artikel in diesem Heft

  1. Frontmatter
  2. Short Communication
  3. Acetone-water mixture is a competent solvent to extract phenolics and antioxidants from four organs of Eucalyptus camaldulensis
  4. Research Articles
  5. Proteases from Calotropis gigantea stem, leaf and calli as milk coagulant source
  6. A new method to quantify atmospheric Poaceae pollen DNA based on the trnT-F cpDNA region
  7. Expression of a functional recombinant vascular endothelial growth factor 165 (VEGF165) in Arabidopsis thaliana
  8. Computational exploration of antiviral activity of phytochemicals against NS2B/NS3 proteases from dengue virus
  9. Investigation of antioxidant, cytotoxic, tyrosinase inhibitory activities, and phenolic profiles of green, white, and black teas
  10. DFR and PAL gene transcription and their correlation with anthocyanin accumulation in Rhodomyrtus tomentosa (Aiton.) Hassk.
  11. Comparison of phenolic profiles and antioxidant activity of three Ornithogalum L. species
  12. Increasing the fermentation efficiency of Lactobacillus paracasei ssp. paracasei MIUG BL6 in a rye flour sourdough
  13. Determination of chemical composition, antibacterial and antioxidant properties of products obtained from carob and honey locust
  14. Chitinolytic Bacillus subtilis Ege-B-1.19 as a biocontrol agent against mycotoxigenic and phytopathogenic fungi
  15. Recycling fish skin for utilization in food industry as an effective emulsifier and foam stabilizing agent
  16. A novel, thermotolerant, extracellular PHB depolymerase producer Paenibacillus alvei PHB28 for bioremediation of biodegradable plastics
  17. Post-transcriptional regulation of miRNA-15a and miRNA-15b on VEGFR gene and deer antler cell proliferation
  18. Comparison of pendimethalin binding properties of serum albumins from various mammalian species
  19. Crocin (active constituent of saffron) improves CCl4-induced liver damage by modulating oxidative stress in rats
  20. Time dependent change of ethanol consumption biomarkers, ethyl glucuronide and ethyl sulphate, after single dose ethanol intake
  21. GC-MS analysis and biological activities of Thymus vulgaris and Mentha arvensis essential oil
  22. Immobilization and some application of α-amylase purified from Rhizoctonia solani AG-4 strain ZB-34
  23. Letter to the Editor
  24. Molecular crosstalk between Hog1 and calcium/CaM signaling
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